1. Field of Invention
The present invention relates generally to the synthesis of fluorescent compounds that are analogues of xanthene dyes. The compounds of the invention are fluorophores that are derivatized to allow their facile attachment to a carrier molecule.
2. Background
There is a continuous and expanding need for rapid, highly specific methods of detecting and quantifying chemical, biochemical and biological substances as analytes in research and diagnostic mixtures. Of particular value are methods for measuring small quantities of nucleic acids, peptides, saccharides, pharmaceuticals, metabolites, microorganisms and other materials of diagnostic value. Examples of such materials include narcotics and poisons, drugs administered for therapeutic purposes, hormones, pathogenic microorganisms and viruses, peptides, e.g., antibodies and enzymes, and nucleic acids, particularly those implicated in disease states.
The presence of a particular analyte can often be determined by binding methods that exploit the high degree of specificity that characterizes many biochemical and biological systems. Frequently used methods are based on, for example, antigen-antibody systems, nucleic acid hybridization techniques and protein-ligand systems. In these methods, the existence of a complex of diagnostic value is typically indicated by the presence or absence of an observable “label” which is attached to one or more of the interacting materials. The specific labeling method chosen often dictates the usefulness and versatility of a particular system for detecting an analyte of interest. Preferred labels are inexpensive, safe, and capable of being attached efficiently to a wide variety of chemical, biochemical, and biological materials without significantly altering the important binding characteristics of biological materials without significantly altering the important binding characteristics of those materials. The label should give a highly characteristic signal, and should be rarely, and preferably never, found in nature. The label should be stable and detectable in aqueous systems over periods of time ranging up to months. Detection of the label is preferably rapid, sensitive, and reproducible without the need for expensive, specialized facilities or the need for special precautions to protect personnel. Quantification of the label is preferably relatively independent of variables such as temperature and the composition of the mixture to be assayed.
A wide variety of labels have been developed, each with particular advantages and disadvantages. For example, radioactive labels are quite versatile, and can be detected at very low concentrations, such labels are, however, expensive, hazardous, and their use requires sophisticated equipment and trained personnel. Thus, there is wide interest in non-radioactive labels, particularly in labels that are observable by spectrophotometric, spin resonance and luminescence techniques, and reactive materials, such as enzymes that produce such molecules.
Labels that are detectable using fluorescence spectroscopy are of particular interest, because of the large number of such labels that are known in the art. Moreover, as discussed below, the literature is replete with syntheses of fluorescent labels that are derivatized to allow their attachment to other molecules and many such fluorescent labels are commercially available.
In addition to being directly detected, many fluorescent labels operate to quench the fluorescence of an adjacent second fluorescent label. Because of its dependence on the distance and the magnitude of the interaction between the quencher and the fluorophore, the quenching of a fluorescent species provides a sensitive probe of molecular conformation and binding, as well as or other interactions. An excellent example of the use of fluorescent reporter quencher pairs is found in the detection and analysis of nucleic acids.
Fluorescent nucleic acid probes are important tools for genetic analysis, in both genomic research and development, and in clinical medicine. As information from the Human Genome Project accumulates, the level of genetic interrogation mediated by fluorescent probes will expand enormously. One particularly useful class of fluorescent probes includes self-quenching probes, also known as fluorescence energy transfer probes, or FET probes. The design of different probes using this motif may vary in detail. In an exemplary FET probe, both a fluorophore and a quencher are tethered to a nucleic acid. The probe is configured such that the fluorophore is proximate to the quencher and the probe produces a signal only as a result of its hybridization to an intended target. Despite the limited availability of FET probes, techniques incorporating their use are rapidly displacing alternative methods.
Probes containing a fluorophore-quencher pair have been developed for nucleic acid hybridization assays where the probe forms a hairpin structure, i.e., where the probe hybridizes to itself to form a loop such that the quencher molecule is brought into proximity with the reporter molecule in the absence of a complementary nucleic acid sequence to prevent the formation of the hairpin structure (see, for example, WO 90/03446; European Patent Application No. 0 601 889 A2). When a complementary target sequence is present, hybridization of the probe to the complementary target sequence disrupts the hairpin structure and causes the probe to adopt a conformation where the quencher molecule is no longer close enough to the reporter molecule to quench the reporter molecule. As a result, the probes provide an increased fluorescence signal when hybridized to a target sequence than when they are unhybridized.
Assays have also been developed for detecting a selected nucleic acid sequence and for identifying the presence of a hairpin structure using two separate probes, one containing a reporter molecule and the other a quencher molecule (see, Meringue, et al., Nucleic Acids Research, 22: 920-928 (1994)). In these assays, the fluorescence signal of the reporter molecule decreases when hybridized to the target sequence due to the quencher molecule being brought into proximity with the reporter molecule.
One particularly important application for probes including a reporter-quencher molecule pair is their use in nucleic acid amplification reactions, such as polymerase chain reactions (PCR), to detect the presence and amplification of a target nucleic acid sequence. In general, nucleic acid amplification techniques have opened broad new approaches to genetic testing and DNA analysis (see, for example, Arnheim et al. Ann. Rev. Biochem., 61: 131-156 (1992)). PCR, in particular, has become a research tool of major importance with applications in, for example, cloning, analysis of genetic expression, DNA sequencing, genetic mapping and drug discovery (see, Arnheim et al., supra; Gilliland et al., Proc. Natl. Acad. Sci. USA, 87: 2725-2729 (1990); Bevan et al., PCR Methods and Applications, 1: 222-228 (1992); Green et al., PCR Methods and Applications, 1: 77-90 (1991); Blackwell et al., Science, 250: 1104-1110 (1990)).
Commonly used methods for detecting nucleic acid amplification products require that the amplified product be separated from unreacted primers. This is typically achieved either through the use of gel electrophoresis, which separates the amplification product from the primers on the basis of a size differential, or through the immobilization of the product, allowing free primer to be washed away. However, a number of methods for monitoring the amplification process without prior separation of primer have been described; all of them are based on FET, and none of them detect the amplified product directly. Instead, the methods detect some event related to amplification. For that reason, they are accompanied by problems of high background, and are not quantitative, as discussed below.
One method, described in Wang et al. (U.S. Pat. No. 5,348,853; and Anal. Chem., 67: 1197-1203 (1995)), uses an energy transfer system in which energy transfer occurs between two fluorophores on the probe. In this method, detection of the amplified molecule takes place in the amplification reaction vessel, without the need for a separation step.
A second method for detecting an amplification product without prior separation of primer and product is the 5′-nuclease PCR assay (also referred to as the TaqMan™ assay) (Holland et al., Proc. Natl. Acad. Sci. USA, 88: 7276-7280 (1991); Lee et al., Nucleic Acids Res., 21: 3761-3766 (1993)). This assay detects the accumulation of a specific PCR product by hybridization and cleavage of a doubly labeled fluorogenic probe (the “TaqMan” probe) during the amplification reaction. The fluorogenic probe consists of a nucleic acid labeled with both a fluorescent reporter dye and a quencher dye. During PCR, this probe is cleaved by the 5′-exonuclease activity of DNA polymerase if, and only if, it hybridizes to the segment being amplified. Cleavage of the probe generates an increase in the fluorescence intensity of the reporter dye.
Yet another method of detecting amplification products that relies on the use of energy transfer is the “beacon probe” method described by Tyagi et al. (Nature Biotech., 14: 303-309 (1996)) which is also the subject of U.S. Pat. No. 5,312,728 to Lizardi et al. This method employs nucleic acid hybridization probes that can form hairpin structures. On one end of the hybridization probe (either the 5′- or 3′-end) there is a donor fluorophore, and on the other end, an acceptor moiety. In this method, the acceptor moiety is a quencher, absorbing energy from the donor. Thus when the beacon is in the open conformation, the fluorescence of the donor fluorophore is detectable, whereas when the beacon is in hairpin (closed) conformation, the fluorescence of the donor fluorophore is quenched. When employed in PCR, the molecular beacon probe, which hybridizes to one of the strands of the PCR product, is in “open conformation,” and fluorescence is detected, while those that remain unhybridized will not fluoresce. As a result, the amount of fluorescence will increase as the amount of PCR product increases, and thus can be used as a measure of the progress of the PCR.
The probes discussed above are most generally configured such that the quencher and fluorophore are on the 3′- and 5′-ends of the probe (Lyamichev et al., Science, 260: 778-783 (1993)). This spacing of the flurophore and quencher may impede fluorescent energy transfer: fluorescence energy transfer decreases as the inverse sixth power of the distance between the flurophore and quencher. Thus, if the quencher is not close enough to the reporter to achieve efficient quenching the background emissions from the probe can be quite high.
For the xanthene dye to be useful as a label it must posses a chemical functional group the will permit it to bind to, or react with, a substrate of interest. The incorporation of such reactive chemical functionality into xanthene dyes usually requires additional synthetic steps and/or difficult to implement purification methods. In particular, separation of structural isomers of fluoresceins and rhodamines, which are the most commonly used xanthene labeling reagents for biological and medical applications, is tedious and is to be avoided if possible.
The core chemical structure of many fluorescein and rhodamine dyes includes a carboxylic acid group in the ortho position of the benzene ring attached to the xanthene residue; some others posses a sulfonic acid group at this site. The carboxylic acid group has not been widely utilized as a site for conjugation of the dye to substrates due to its low reactivity and due to side reactions that render the dye non-fluorescent. Although the carboxylic acid can be activated and reacted with alcohols to form esters or with amines to form amides, the ester linkage is of insufficient stability to be useful when preparing compounds that are stably labeled with a fluorophore.
The amide linkage is stable to hydrolysis but while some amides prepared from the activated carboxylic acid and primary amines are reported to be colored (Mayer et al., U.S. Pat. No. 4,647,675) others are reported to undergo a spirolactamization reaction in which the dye loses its color and is rendered non-fluorescent (Adamczyk et al., Synthetic Commun. 31: 2681-2690 (2001); and Cincotta et al., U.S. Pat. No. 4,290,955)). In contrast, secondary amines react with the activated carboxylic acid to create an amide link that cannot undergo spirolactamization, providing a xanthene dye that retains its color and fluorescence (Gao et al., WO 02/055512). Menchen et al. disclose xanthene dyes in which the ortho carboxyl moiety is activated and coupled to another species. Other amide derivatized xanthene dyes are disclosed in Haugland et al., U.S. Pat. No. 6,399,392; and Mayer et al., U.S. Pat. No. 4,935,059.
Xanthene dyes that posses a sulfonic acid group in the ortho position can be activated and reacted with alcohols and with amines in a manner similar to xanthene dyes with ortho carboxylic acid groups to yield sulfonate esters and sulfonamides, respectively. The sulfonate esters are not stable under aqueous conditions and are of little use as linker functionality for preparing oligonucleotides. The sulfonamides are stable and have been used to prepare reactive xanthene dyes such as succinimidyl esters, maleimides and phosphoramidites.
None of the above-described references discloses or suggests the modification of the fluorophore nucleus with a versatile amide-linked moiety that allows for the facile variation of the composition, length and degree of branching of the linker. Furthermore, none of the references suggest a linker that provides a locus for attaching the fluorophore to a solid support, nor do the references describe a branched linker moiety that tethers both a phosphoramidite and a dimethoxytrityl ether to a single endocyclic nitrogen atom.
Attaching quenchers or fluorophores to sites other than the readily accessible 5′-OH group generally requires the synthesis of fluorescent labels that are of use to attach the fluorophore to a single reactive residue of a carrier molecule or a selected reactive functional group on that residue; reacting the same fluorophore with a different functional group of the carrier generally requires a new modification of the fluorescent core. Similarly, modifying the structure or composition of the linker arm requires a modification to the fluorophore nucleus. Thus, a xanthene label that provides a versatile entry point for an array of synthetic modifications would represent a significant advance in the art.